Throughout the life of an organism, there is a constant progression of cellular development. New cells are continually being produced, through such mechanisms as mitosis and meiosis, to replace old or damaged cells that are targeted for destruction due to disease, injury, extracellular cues and internal instructions. This cellular life-death balance is a critical feature not only in normal animal development, but also in pathogenesis. Indeed, diseases such as cancer and autoimmune disorders are associated with decreased cell death, whereas AIDS and neurodegenerative disorders are associated with increased cell death (Thompson, 1995).
Programmed cell death, or apoptosis, is one manner in which cells that are no longer needed or that no longer function normally can be eliminated. Apoptosis is believed to be a process actively regulated by the environment in which cells live. This process is critical to the normal development of all multicellular organisms and to the maintenance of homeostasis within such organisms (Raff, 1992). Moreover, apoptosis is vital in the defense against viral infection and in preventing the development of carcinogenesis. Amongst multicellular organisms, the apoptotic pathway leading to cell death is highly conserved (Hengartner, 1994).
Many molecules that regulate the apoptotic pathway have been identified, including both positive regulators (agonists) and negative regulators (antagonists). Such regulators are often members of the same family of polypeptides, and can have roles important in the extracellular, cell surface, and/or intracellular steps of the apoptotic pathway (Oltvai and Korsmeyer, 1994). One such family of polypeptides, constituting an intracellular checkpoint in the apoptotic pathway, is the Bcl-2 family of polypeptides (“Bcl-2 family”). This important family of apoptotic regulators can be divided into two classes: those that suppress cell death (apoptotic antagonists) (e.g., Bcl-2, Bcl-XL, MCL-1, and A1) and those that appear to promote apoptosis (apoptotic agonists) (e.g., BAX, BAK, Bcl-XS, and BAD). The first member of the Bcl-2 family to be identified was Bcl-2, a cell death inhibitor encoded by the bcl-2 proto-oncogene, initially isolated from cells of a follicular lymphoma (Bakhshi et al., 1985; Tsujimoto et al., 1985; Cleary and Sklar, 1985). Bcl-2 is a 26 kD integral membrane polypeptide localized to the mitochondria that extends or promotes the survival of many different cell types by inhibiting apoptosis induced by a variety of cell death-inducing stimuli (Korsmeyer, 1992).
The Bcl-2 family contains members that are structurally and functionally related to Bcl-2, and is defined by polypeptides having amino acid sequence homology to one or more of four conserved motifs, termed Bcl-homology (BH1, BH2, BH3, and BH4) domains (for reviews see Reed, 1997 and Chittenden, 1998). The Bcl-homology domains have been shown to be important in the formation of homodimers and heterodimers, both among and between Bcl-2 family members.
The functional characteristics of these proteins vary, depending on their dimerization partners (Yin et al., 1994; Boyd et al., 1995; Chittenden et al., 1995; Farrow and Brown, 1996). The dimerization status of the proteins has been shown to depend on the intracellular concentrations of the particular family members, and is directly related to whether a cell will respond to an apoptotic signal (Oltvai and Korsmeyer, 1994). Moreover, the formation of dimers between cell death promoters and cell death inhibitors is competitive. For example, both Bcl-2 and Bcl-XL enhance cellular survival, while BAD and BAX promote cell death (Oltvai et al., 1993). However, when BAD is overexpressed, it counters the death inhibitory activity of Bcl-XL, and to a lesser extent Bcl-2, by the formation of heterodimers. It is thought that BAD competes with BAX for binding to Bcl-XL. Therefore, when the intracellular levels of BAD increase, there is a sequestration of Bcl-XL in BAD:Bcl-XL heterodimers, and a concomitant increase in the amount of free BAX present in the cell as BAX monomers or BAX:BAX homodimers. The increased levels of BAX monomer and homodimers, in turn, promote cellular susceptibility to apoptosis (Korsmeyer, U.S. Pat. No. 5,856,445).
BAD (Bcl-XL/Bcl-2 Associated Cell Death Regulator polypeptide) is a cell death promoter distantly related to Bcl-2. It has been sequenced and shown to share identity with Bcl-2 only within the BH3 domain. BAD is an unique pro-apoptotic member of the Bcl-2 family in that its function is regulated by phosphorylation (Yang et al., 1995), suggesting an important connection between extracellular apoptosis regulatory agents, intracellular signaling pathways and the function of this Bcl-2 family member. As discussed above, BAD is believed to play a role in the apoptotic signaling pathway through an association with Bcl-2 family members, chiefly the cell death inhibitors Bcl-XL and Bcl-2 (Yang et al., 1995). Upon being dephosphorylated, BAD is active and forms heterodimers with Bcl-XL and Bcl-2, thereby displacing BAX and promoting cell death. The death-promoting activity of BAD can be inhibited by the phosphorylation of either of two serine residues, corresponding to the serines at position 112 and position 136 in the amino acid sequence of murine BAD (SEQ ID NO:2). Upon phosphorylation of either of these two sites, BAD no longer binds Bcl-XL or Bcl-2 and instead, is thought to be bound by the phosphoserine-binding protein 14-3-3, thereby allowing Bcl-XL and Bcl-2 to perform their anti-apoptotic functions (Zha et al., 1996).
The interaction of murine BAD and the cytosol polypeptide 14-3-3 was discovered using a GST-BAD fusion polypeptide having a heart muscle kinase (HMK) motif. The fusion polypeptide was labeled with γ32P-ATP in vitro and used as a probe to screen an oligo (dT)-primed day-16 mouse embryonic EXlox cDNA expression library (Blanar and Rutter, 1992). Thereby two independent clones, each encoding a polypeptide of the tau form (τ) of 14-3-3 that bound to the BAD fusion polypeptide, were isolated (Nielsen, 1991).
The members of the 14-3-3 family, identified in at least seven mammalian isoforms, are highly conserved and ubiquitously expressed. They bind to and regulate a variety of proteins, including a number of proteins involved in signal transduction. Family members recognize and bind sequences containing a conserved phosphoserine motif (Muslin et al, 1996).
Muslin et al. identified a number of polypeptides, including BAD, that contain this motif and postulated that if these other polypeptides were appropriately phosphorylated, 14-3-3 would bind them as well. However, they did not perform any experiments to determine whether 14-3-3 in fact binds phosphorylated BAD, nor did they discuss possible physiological consequences of such binding. Moreover, there was no discussion of other potential BAD serine phosphorylation sites. Only the general suggestion that 14-3-3 might interact with polypeptides to perform an essential chaperone function was advanced. Korsmeyer also suggested a likely role for 14-3-3 as a chaperone or protective binding polypeptide. In the case of BAD, it was suggested that 14-3-3 might facilitate the translocation of phosphorylated BAD from the mitochondrial membrane to cytosolic compartments, sequestering it therein (U.S. Pat. No. 5,856,445).
Thus far, BAD is the only known pro-apoptotic member of the Bcl-2 family whose function is regulated by phosphorylation. The serine/threonine kinase Akt, a downstream effector of PI 3-kinase, phosphorylates murine BAD on the serine at position 136 (also called “Ser136” or “serine-136”), thereby preventing murine BAD from associating with Bcl-2 or Bcl-XL, and freeing these proteins to promote cell survival. (Datta et al., 1997; del Peso et al., 1997).
In addition, murine BAD is phosphorylated at the serine at position 112 (also called “Ser112” or “serine-112”). Although phosphorylation of Ser136 was sufficient to prevent BAD from binding to Bcl-XL, phosphorylation of Ser112 appeared critical for cellular survival in some cell types but not in others (Zha et al., 1996; Datta et al., 1997). Several candidate enzymes have been proposed to be responsible for the phosphorylation of Ser112, including PKA, c-Raf, and MEK (Harada et al., 1999; Wang and Reed, 1998; Scheid and Duronio, 1998). However, the discoveries of the present invention indicate that Ser112 is, at best, a minor site of phosphorylation by PKA. Similarly, Akt does not appear to phosphorylate BAD on Ser112.
Some disease conditions may be related to the development of a defective down-regulation (i.e., inhibition or modulation) of apoptosis in the affected cells. For example, neoplasias may result, at least in part, from an apoptosis-resistant state in which cell proliferation signals inappropriately exceed cell death signals and apoptosis is thereby down-regulated. Furthermore, some DNA viruses such as Epstein-Barr virus, African swine fever virus and adenovirus, parasitize the host's cellular machinery to drive their own replication and at the same time inhibit or modulate apoptosis, thereby repressing cell death and allowing the host cell to continue reproducing the virus. Moreover, certain other disease conditions such as lymphoproliferative conditions, arthritis, inflammation, autoimmune diseases, and cancers, including drug-resistant cancers, may result from a down-regulation (e.g., inhibition or modulation) of apoptosis. In such disease conditions it would be desirable to promote or induce apoptosis. By manipulating members of the signal transduction cascade that trigger apoptosis, one could selectively induce apoptosis. For example, BAD could be altered to promote or induce its binding to Bcl-XL and/or Bcl-2 and, thereby, diminish (or inhibit or modulate) the cell-survival promoting activity of these cell death inhibitors.
Conversely, in certain disease conditions it would be desirable to inhibit or modulate apoptosis, for example, in the treatment of immunodeficiency diseases, including AIDS, senescence, neurodegenerative disease, ischemic and reperfusion cell death, infertility, and wound-healing. In such cases, BAD could be altered to block its ability to bind Bcl-XL and/or Bcl-2 and, thereby, promote or induce the cell death repressor, or anti-apoptotic, activity of these cell death inhibitors.
Accordingly, it would be desirable to identify novel compositions and methods that could be used to modulate the binding of BAD to members of the Bcl-2 family, such as Bcl-XL and Bcl-2, and thereby induce, promote, inhibit or modulate apoptosis, or induce, promote, inhibit, or modulate cell survival, and to utilize these novel compositions and methods as a basis for treatment of disease conditions involving either inappropriate inhibition or inappropriate acceleration of cell death.